PROJECT SUMMARY Current treatments for Mycobacterium tuberculosis infections suffer from complicated regimens leading to patient non-compliance, increasing incidences of antibiotic-resistant strains, and inefficacy against non- replicating bacteria in granulomas that cause latent stages of disease. The urgency to develop new tuberculosis therapeutics is highlighted by the fact that ~25% of people globally are infected with asymptomatic latent disease, and over 10 million cases of active tuberculosis infection are reported annually, with 1.6 million deaths attributed to the disease. To address this dilemma, our strategy is to develop new antibiotic classes that exploit unique biological targets, specifically bacterial GroEL/ES chaperonin systems. The primary function of GroEL/ES chaperonin systems are to fold newly translated proteins into their functional conformations. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation where non-replicating bacteria contribute to latent disease, while GroEL2 is essential and likely functions as the canonical housekeeping chaperonin for folding proteins. Thus, our central hypothesis is that inhibitors that can target the two GroEL homologs in M. tuberculosis will be effective against both actively- replicating bacteria and preventing bacteria from forming and residing in granulomas in a non-replicating state. Supporting this hypothesis, our recent studies have indicated that bioactive molecules, in particular those with antibiotic properties, may have a significant predisposition for inhibiting GroEL/ES chaperonin systems. Furthermore, we also identified GroEL/ES inhibitors that exhibited moderate antibiotic effects against actively replicating M. tuberculosis, and conversely found that bedaquiline and delamanid were weak GroEL/ES inhibitors. Towards our long-term goal of developing such mechanistically unique antibiotic candidates, our objective in the proposed study is to identify new hit-to-lead antibiotic candidates that are more potent at inhibiting GroEL/ES chaperonin systems and killing actively replicating and non-replicating M. tuberculosis. We will take a two-pronged approach to accomplish this objective. In Aim 1, we will screen our larger library of known GroEL/ES inhibitors to identify new scaffolds with greater potency against actively-replicating and non- replicating M. tuberculosis. In Aim 2, we will screen a library of known M. tuberculosis inhibitors to identify hits that can target GroEL/ES chaperonin systems. The proposed study will have significant impact for developing new therapeutic candidates with efficacy against all stages of tuberculosis, which would be a significant advance to augment current therapeutics that primarily target actively-replicating bacteria.